U.S. patent number 4,997,915 [Application Number 07/333,964] was granted by the patent office on 1991-03-05 for purification of pertussis toxins.
This patent grant is currently assigned to Connaught Laboratories Limited. Invention is credited to Dirk Alkema, Gail Jackson, Larry U. L. Tan, Po S. Wah.
United States Patent |
4,997,915 |
Tan , et al. |
March 5, 1991 |
Purification of pertussis toxins
Abstract
Lymphocytosis promoting factor (LPF) and filamentous
hemaglutinin (FHA) are isolated from the growth medium of the
Bordatella pertussis organism and purified by selecting adsorbing
the LPF and FHA on a selective adsorbing medium, such as filter
aids or gel filtration media, at low ionic strength and
subsequently removing the adsorbed LPF and FHA at using an aqueous
medium of high ionic strength, either simultaneously or
sequentially. Prior to desorbtion of the LPF and FHA, the adsorbing
medium may be contacted with an aqueous solution of a non-ionic
detergent, which enables the LPF and FHA subsequently desorbed to
be substantially free from contamination by lipopolysaccharides
(LPS). The LPF and FHA may be further purified on hydroxyapatite.
The LPF and FHA may be detoxified separately or together by
contacting with a cross-linking agent, such as glutaraldehyde or
formaldehyde, in the presence of an anti-aggregation agent. The
resulting purified and detoxified LPF and FHA may be used to
formulate a vaccine against pertussis.
Inventors: |
Tan; Larry U. L. (Mississauga,
CA), Alkema; Dirk (Stayner, CA), Jackson;
Gail (Richmond Hill, CA), Wah; Po S. (Willowdale,
CA) |
Assignee: |
Connaught Laboratories Limited
(Willowdale, CA)
|
Family
ID: |
10634535 |
Appl.
No.: |
07/333,964 |
Filed: |
April 6, 1989 |
Foreign Application Priority Data
Current U.S.
Class: |
530/396;
435/71.2; 530/380; 424/254.1 |
Current CPC
Class: |
A61P
31/04 (20180101); C07K 14/235 (20130101); A61K
39/00 (20130101) |
Current International
Class: |
C07K
14/235 (20060101); C07K 14/195 (20060101); A61K
39/00 (20060101); A61K 039/02 (); A61K 039/10 ();
C07K 003/18 (); C07K 003/12 () |
Field of
Search: |
;424/92 ;530/380,396
;435/71.2 |
References Cited
[Referenced By]
U.S. Patent Documents
|
|
|
4455297 |
June 1984 |
Syukuda et al. |
4563303 |
January 1986 |
Ginnaga et al. |
4687738 |
August 1987 |
Ginnaga et al. |
4705686 |
November 1987 |
Scott et al. |
4784589 |
November 1988 |
Robinson et al. |
|
Primary Examiner: Schain; Howard E.
Assistant Examiner: Koh; Choon P.
Attorney, Agent or Firm: Sim & McBurney
Claims
What we claim is:
1. A method for the isolation and purification of the proteinaceous
materials called lymphocytosis promoting factor (LPF) and
filamentous hemaglutinin (FHA) from a growth medium in which has
been grown the Bordatella pertussis organism, which comprises:
contacting said growth medium at an ionic strength corresponding to
a conductivity of about 11 mS/cm or less with a solid particulate
adsorbing medium to selectively adsorbing LPF and FHA from the
growth medium, and
sequentially or simultaneously desorbing the selectively adsorbed
proteinaceous materials by contacting said adsorbing medium with an
aqueous medium of ionic strength corresponding to a conductivity of
greater than about 11 mS/cm and sufficient to effect said
desorption.
2. The method of claim 1 wherein said adsorbing medium is selected
from the group consisting of filter aids, siliceous materials,
celluloses, agaroses and gel filtration media.
3. The method of claim 1, wherein said adsorbing medium is selected
from Perlite and Celite.
4. The method of claim 1, wherein said growth medium has an ionic
strength of about 4 mS/cm or less when contacting said adsorbing
medium to effect said selected adsorption.
5. The method of claim 4, wherein said aqueous medium has an ionic
strength of at least about 50 mS/cm when contacting said adsorbing
medium to effect said desorption.
6. The method of claim 1, wherein said FHA and LPF are desorbed
from said adsorbing medium simultaneously.
7. The method of claim 6, wherein said simultaneously desorbed LPF
and FHA are subsequently separated one from another.
8. The method of claim 1, wherein said adsorbing medium first is
contacted with an aqueous medium having an ionic strength
sufficient to selectively desorb LPF in preference to said FHA and
then is contacted with an aqueous medium having an ionic strength
sufficient to desorb FHA.
9. The method of claim 8, wherein said first-mentioned aqueous
medium has an ionic strength from about 11 mS/cm to about 20 mS/cm
and said second-mentioned aqueous medium has an ionic strength of
at least about 20 mS/cm.
10. The method of claim 1, wherein, subsequent to said adsorption
step and prior to said desorption step, said adsorbing medium is
contacted with an aqueous non-ionic detergent solution selective to
elute lipopolysaccharides (LPS) from the adsorbing medium in
preference to said LPF and FHA.
11. The method of claim 10, wherein said non-ionic detergent
solution is effective to decrease the LPS contamination of said LPF
and FHA after said desorbtion to about 1 to about 10 ng/ml.
12. The method of claim 11 wherein said non-ionic detergent is
selected from Triton X-100 of concentration of 0.005 to about 5%
(v/v) and Nonidet p40 in a concentration of about 0.0005 to about
0.1% (v/v).
13. The method of claim 11, wherein, subsequent to desorption of
said LPF and FHA, the purified proteinaceous material is detoxified
by contact with a cross-linking agent in the presence of an
anti-aggregation agent.
14. The method of claim 13, wherein said cross-linking agent is
selected from glutaraldehyde, formaldehyde and mixtures thereof and
said anti-aggregation agent is selected from sucrose, glycerol and
mixtures thereof.
15. The method of claim 13 wherein, subsequent to desorption of
said LPF and FHA and prior to said detoxification, said LPF and FHA
are subjected to further purification on hydroxyapatite.
16. The method of claim 15, including the subsequent steps of
sterilizing the proteins and formulating them as a vaccine against
pertussis.
17. The method of claim 1 wherein:
said growth medium has an ionic strength corresponding to a
conductivity of 4mS or less and is contacted with Perlite as said
adsorbing medium to provide an adsorbed protein loading of about 1
to about 5 mg per milliliter of Perlite,
said Perlite is provided in the form of a packed column having a
height of about 15 to about 18 cm and a diameter of about 10 to
about 45 cm and said growth medium is contacted with said Perlite
at a linear flow rate of about 50 to about 200 cm/hr.,
said column having adsorbed LPF and FHA is washed with about 2 to
about 10 column volumes of a buffer containing about 10 to about 50
mM Tris HCl at pH 8.0, then is washed with an aqueous non-ionic
detergent solution to remove lipopolysaccharides (LPS) from the
column and to provide an overall decrease in the LPS/LPF weight
ratio between that in said growth medium and that in eluted LPF of
between about 10,000 and about 100,000, and subsequently is washed
further with said buffer to remove said non-ionic detergent from
said column and to provide a washed column, and
LPF is eluted from the column by contacting said washed column with
an aqueous eluting medium containing about 0.1 to about 0.2 mM
sodium chloride therein and said FHA is subsequently eluted from
the column by contacting said washed column with an aqueous eluting
medium containing at least 0.2M sodium chloride therein.
18. The method of claim 17 wherein the eluate containing LPF is
further purified by:
contacting a column of hydroxyapatite having a diameter of about 5
to about 30 cm and a height of about 5 to about 8 cm with said
eluate at a loading of about 0.5 to about 1 mg of protein/ml of
hydroxyapatite at a linear flow rate of about 15 to about 25 cm/hr
to adsorb said LPF thereon, and
subsequently eluting the LPF from said hydroxyapatite using an
aqueous eluant medium containing about 0.1 to about 0.3M sodium
chloride.
19. The method of claim 17 wherein the eluate containing FHA is
further purified by:
contacting a column of hydroxyapatite having a diameter of about 5
to about 30 cm and a height of about 5 to about 8 cm with said
eluate at a loading of about 0.5 to about 1 mg of protein/ml of
hydroxyapatite at a linear flow rate of about 15 to about 25 cm/hr
to adsorb said LPF thereon, and
subsequently eluting the FHA from said hydroxyapatite using an
aqueous eluant medium containing at least about 0.2M sodium
chloride.
Description
FIELD OF INVENTION
The present invention relates to a novel method of isolation and
purification of specific proteins from the fermentation broth of
the organism Bordatella pertussis, the removal of pyrogenic factors
from these proteins, the detoxification of the proteins and the
preparation from these purified proteins of a vaccine that is
virtually non-toxic, has little or no side effects and confers
protection against the disease of pertussis.
BACKGROUND OF THE INVENTION
For infants below the age of 24 months, the disease caused by
Bordatella pertussis, pertussis or whooping cough, can be very
severe and has a mortality rate of approximately 1%. Over the last
fifty years, three types of vaccine have been available for
immunization against the disease. The most widely used vaccine,
that has been available for the protection of infants against
pertussis infection, in combination with tetanus and diphtheria
vaccines, is the so-called "whole cell" vaccine, which is available
in all developed countries, and many of the Third World
countries.
This whole cell pertussis vaccine is prepared by growing known
strains of the B.pertussis organism in a defined medium for several
hours in a fermentor, until the mixture reaches certain defined
parameters. The mixture then is treated with a chemical agent, such
as formaldehyde, which kills the organism and detoxifies proteins
present in the supernatant and in the organism itself. After
allowing the mixture to stand for a specified time to ensure that
this detoxification procedure is complete, the cells are separated
from the supernatant by passing the mixture through a continuous
centrifuge, to provide a packed mass of cells and the supernatant,
which is discarded. The cells then are resuspended in a solution of
sodium chloride to provide a suspension, that, when diluted to a
known strength, usually determined by the opacity of the
suspension, and injected subcutaneously, elicits antibodies that
are protective against the disease. This "whole cell" vaccine is
known for giving minor local reactions at the injection site with
occasionally more severe overall reactions, such as elevated
temperature and general fretfulness. There has been speculation
that the vaccine is responsible for some neurological reactions in
infants.
In a second vaccine type, the B.pertussis was grown and detoxified
with formaldehyde as before, and then the isolated cells extracted
with a concentrated solution of urea. After filtration and dialysis
to remove the urea, a mixture of soluble cell wall components is
obtained, which, after dilution to a known strength, was in use as
a vaccine from 1969 to 1974, but has now been withdrawn because of
poor efficacy. More recently, a third vaccine type, commonly called
an acellular vaccine, that is in use in Japan and is in clinical
trials in a number of other developed countries, is prepared by
isolation and purification of constituents of the culture
supernatants of B.pertussis after detoxification. Specifically, the
constituents called lymphocytosis promoting factor (LPF) also known
as pertussis toxin (PT), filamentous hemaglutinin (FHA) and
agglutinogens have been isolated and identified. However, because
of variations in growth of the organism and the method of
isolation, which is non-specific, the composition of the isolated
mixture of proteins can vary. At present there is no vaccine in
general use and licensed that is completely non-toxic and still
gives good protection.
With the growth of the science of immunology over the years, it has
been recognized that protective antibodies against a specific
disease can be elicited by the administration of specific cellular
components of the organism that causes the disease, rather than the
whole organism that has been inactivated or has been attenuated to
give a non-pathogenic strain. It has been recognized that use of
detoxified or attenuated organisms as a vaccine can introduce
components that may be damaging to the recipient. With this in
mind, a number of efforts have been made to isolate components of
the pertussis organism, either from the cell or that have been
excreted into the medium, that could have antigenic capabilities,
be completely non-toxic and thus could act as vaccines.
As yet there has been no convincing proof that any one particular
cell component, by itself, can act as a protective antigen.
However, in a number of publications and Patent applications (see,
for instance, European Patent Application Nos. 0231083 and 0175841)
it has been suggested that a mixture of the purified and detoxified
proteins, lymphocytosis promoting factor (LPF) and filamentous
hemaglutinin (FHA), can act as a combined antigen and, when
administered to a mammal, generate antibodies that confer
protection against the disease. In the aforesaid publications
various methods have been disclosed for obtaining these proteins in
a purified form, and once purified using them in varying
proportions as a vaccine.
Although existing technology produces highly purified LPF and FHA,
the processes have inherent drawbacks. The affinity chromatographic
methods that have been used are effective but conditions of
absorption and elution often use materials which are toxic,
expensive and/or denature the required proteins. In addition, some
of the materials used on affinity columns can be leached into the
product under the harsh elution conditions required and, since some
of the leached materials are blood derived, may introduce the
possibility of blood born diseases or autosensitization. The use of
gel filtration materials and hydroxyapatite are acceptable when
used by themselves, but give only low purification factors.
The LPF and FHA, which are produced by B.pertussis, also represent
a major challenge in the removal of lipopolysaccharides (LPS) as a
contaminant. LPS even in nanogram quantities can produce fever and
are an undesirable component of any vaccine. The initial
concentration of LPS in the fermentation supernatant can be as high
as one milligram per milliliter. A number of methods have been used
to remove pyrogens from vaccines (see, for instance, U.S. Pat. Nos.
4,000,257 and 4,380,511). Many of these methods are too harsh and
result in denaturation of the required proteins. Other methods are
ineffective, cumbersome to use and expensive.
Before using the LPF and FHA in a vaccine, these proteins must be
detoxified since the LPF in its natural state is highly toxic and
small amounts are still present in the purified FHA. This process
previously has been achieved by treating the proteins with a
chemical agent which induces cross-linking. Traditionally, the
agents used have been formaldehyde and glutaraldehyde. The use of
formaldehyde and glutaraldehyde can lead to heavy losses due to
aggregation and precipitation.
SUMMARY OF INVENTION
In accordance with the present invention, there is provided a novel
method for the isolation and purification of the proteinaceous
materials LPF and FHA from the growth medium of B.pertussis by
adsorption and desorption on various substrates, using a
combination of low and high ionic strength solutions. In addition,
there is provided an improved method of removing pyrogenic factors,
as exemplified by LPS, from the LPF and FHA by washing the adsorbed
proteins with a detergent solution. Further, there is provided an
improved method for the detoxification of the LPF and FHA, using a
cross-linking agent in the presence of an anti-aggregation agent,
such that the purified materials can be readily combined into an
efficacious vaccine for the prevention of the disease of
pertussis.
Accordingly, in one aspect of the present invention, there is
provided a method for the isolation and purification of the
proteinaceous materials called lymphocytosis promoting factor (LPF)
and filamentous hemaglutinin (FHA) from a growth medium in which
has been grown the Bordatella pertussis organism. The method
comprises contacting the growth medium at low ionic strength with a
solid particulate adsorbing medium to selectively adsorb LPF and
FHA from the growth medium, and sequentially or simultaneously
desorbing the proteinaceous materials by contacting the adsorbing
medium with an aqueous medium of high ionic strength.
The isolated LPF and FHA, after further purification and
detoxification, can be formulated into a non-toxic vaccine for
protection against pertussis.
GENERAL DESCRIPTION OF INVENTION
The inventors have determined that LPF and FHA can be adsorbed
preferentially from the filtered growth medium of B.pertussis, at
low ionic strength, onto a variety of solid particulate adsorbent
materials. After the LPF and FHA are adsorbed from the growth
supernatant at low ionic strength onto the substrates, they are
desorbed from the adsorbent material using an aqueous solution of
high ionic strength.
The high ionic strength desorbing medium is an aqueous solution of
a salt and/or buffer. The term "salt solution" used herein refers
to all metal or ammonium salts, such as potassium nitrate, sodium
chloride and ammonium sulfate, which when dissolved in water,
dissociate into their constituent ions, thereby increasing the
ionic strength of the solution without significantly changing the
pH of the solution. The term "buffer" used herein refers to a
chemical compound which, when dissolved in water, dissociates into
their constituent ions, thereby increasing the ionic strength of
the solution and having buffering capacity.
As used herein, the term "low ionic strength", refers to an aqueous
medium having a conductivity of about 11 mS/cm or less, preferably
about 4 mS/cm. The unit of measurement mS/cm is millisiemen per
centimeter. A siemen (S) is a unit of conductivity and is the
equivalent of the inverse of resistance (ohm) and is sometimes
designated mho. The term "high ionic strength" as used herein
refers to an aqueous medium having a conductivity of greater than
about 11 mS/cm and preferably at least about 50 mS/cm.
Solid particulate adsorbent materials useful in the present
invention include filter aids, such as Perlite (which is of
volcanic ash origin) and Celite (a diatomaceous earth), siliceous
materials, such as sand, celluloses, agaroses and gel filtration
materials, such as the Sepharoses, the Sephadexes, ultragel and
their derivatives.
The variety of matrix materials which have been found useful for
the adsorbing medium in the present invention suggests that the
characteristics of the matrix material are non-critical but rather
it is the property of LPF and FHA that, under low ionic strength
conditions, they will bind to a large variety of matrices.
While not wishing to be found by any particular theory to explain
the process of the invention, it is thought that, under the initial
low ionic strength conditions employed, the LPF and FHA are close
to coming out of solution. By passing the solution in contact with
insoluble particulate matrices, the particles of the matrix act as
nuclei onto which the LPF and FHA can precipitate. Resolubilization
for desorbtion then requires a higher ionic strength solution.
After desorption from the absorbing medium by the high ionic
strength solution, a mixture of the two proteins is obtained, that
can be further separated on other materials, such as hydroxyapatite
or other ion-exchange resins, to give the two proteins in high
yields and high purity. Alternatively, we have found that
separation of FHA and LPF after adsorption onto the adsorbing
medium can be obtained by desorbing from the adsorbing medium at
differing ionic strengths. To effect preferential elution of LPF
from the adsorbing medium, an ionic strength of solution of about
11 mS/cm to about 20 mS/cm is employed. Once the LPF has been
eluted, FHA can be eluted at an ionic strength of solution of at
least 20 mS/cm, preferably at least about 50 mS/cm.
The ability to effect adsorption at low ionic strength and
subsequent elution at high ionic strength of LPF and FHA on
conventional gel filtration media and the other non-derivatized
adsorbing media used herein is totally unexpected and deviates from
the state of the art, where proteins are not adsorbed to gel
filtration media and where the protein is continuously eluted from
the column under isocratic, i.e., a single buffer, conditions. The
gel media have been chosen in previous work because of their very
low non-specific protein adsorption, and yet, under the conditions
of the invention, they will still adsorb the LPF and FHA very well.
It has been shown by the inventors that FHA and LPF can be purified
to a greater degree on agarose than derivatized agarose.
The method can be used either as a batch process on the cell free
media obtained from the growth of the organism or as a separation
method on a chromatography column of the adsorbent. Because of
their ease of filtration, their low costs and the accepted
employment of filter-aids in the manufacture of pharmaceutical
products, the use of the filter-aids is preferable to the use of
other materials, such as gel filtration media, and derivatized
materials.
The inventors have further found that, if the proteins adsorbed
onto the matrices are washed, before elution, with an aqueous
non-ionic detergent solution, the LPS in the final product can be
reduced by a factor of 10,000 to 100,000, to a concentration of
about 1 to about 10 ng/mL. Examples of suitable non-ionic detergent
solutions are Triton X-100 in a concentration of about 0.005 to
about 5% (v/v), preferably about 0.1 to about 1% (v/v), and Nonidet
p40 in a concentration of about 0.0005 to about 0.1% (v/v),
preferably about 0.001 to about 0.01% (v/v).
It has also been found by the inventors that the purified LPF and
FHA can be detoxified by contact with a cross-linking agent, such
as glutaraldehyde and/or formaldehyde in the presence of an
anti-aggregation agent, such as glycerol or sucrose, to improve the
yield of final product. The anti-aggregation agent is present in a
significant proportion during the detoxification operation and
prevents the aggregation and precipitation that occurs in the
absence of such material. For the detoxification of LPF in the
presence of glycerol, glycerol is present in an amount of about 30
to about 80% (v/v), preferably approximately 50%, while for the
detoxification of FHA, glycerol is present in an amount of about 10
to about 80% (v/v), preferably approximately 25%. Where sucrose is
used as the anti-aggregation agent, the sucrose is present in an
amount of about 30 to about 60% (w/v), preferably approximately
40%.
In the present invention, B.pertussis is grown in a fermentor using
controlled conditions. Carbon sources and growth factors are
supplemented continuously or in batches at various intervals during
the fermentation until the two proteins, LPF and FHA, are at the
desired level, which can be determined by enzyme linked
immunosorbent assay (ELISA). In our invention, the mixture of cells
and medium from the fermentor is not inactivated by chemical means
immediately after the fermentation is complete, but later in the
purification process. The use of chemical detoxification at this
stage in the process can lead to aggregation of the proteins and
poor separation in the following steps.
The fermentor then is harvested and the majority of the cells
removed by continuous centrifugation. The remainder of the cells
then can be removed from the supernatant by filtration, using known
membrane filters of 0.2 .mu. pore size, which also sterilizes the
solution. In the present invention, it is this supernatant that is
retained and processed for isolation and purification of LPF and
FHA. After centrifugation and filtration, the supernatant is
concentrated, say 10-fold, using membrane filtration, assayed for
protein and then diluted until the ionic strength is in the
required range. This solution, which contains the LPF and FHA
proteins then is treated with the adsorbing medium, either in a
batch process or as a chromatographic process. The LPF and FHA,
after adsorption onto the adsorbing medium, are washed first with
several volumes of a buffer to remove contaminants and then with a
solution of a detergent which removes the majority of the
lipopolysaccharides (LPS). Further washing removes any traces of
the detergent.
The LPF and FHA can be obtained separately from the adsorbent by
elution with solutions of stepwise increasing ionic strength or the
two proteins can be eluted together using a high ionic strength
salt solution. The proteins can be further purified on a
chromatography column of a material, such as hydroxyapatite. The
proteins are eluted from such a column by using different ionic
strengths solutions after prior washing. The separate proteins then
are detoxified in the presence of an anti-aggregation agent to
result in high yields of detoxified protein. After removal of the
additives, the detoxified proteins are sterilized by filtration
and, after assay, can be mixed in the required proportion to give a
solution that can be used as a vaccine against pertussis.
DESCRIPTION OF PREFERRED EMBODIMENT
The procedure of the invention may be employed to effect large
scale separation of LPF and FHA using a chromatographic column of
Perlite, which is currently the best mode known to the applicants
for effecting separation and recovery of purified LPF and FHA.
Concentrated B.pertussis fermentation broth is diluted to a low
ionic strength corresponding to a conductivity of 4mS or less and
loaded onto a column of packed Perlite to provide a protein loading
of about 1 to about 5 mg, preferably about 2 to about 3 mg per
milliliter of packed Perlite. The packed Perlite column usually
about 15 to about 18 cm high and about 10 to about 45 cm in
diameter.
The dilute fermentation broth is contacted with the Perlite column
at a linear flow rate of about 50 to about 200 cm/hr, preferably
approximately 100 cm/hr. The proteins which are adsorbed to the
Perlite are almost exclusively LPF and FHA with most of the
contaminating proteins and LPS passing through the column.
The column then is washed with about 2 to about 10 column volumes
of a buffer containing about 10 to about 50 mM, of Tris HCl at pH
8.0. A subsequent wash with an aqueous non-ionic detergent,
typically about 5 column volumes of an 0.5% (v/v) Triton X-100
solution in 50 mM Tris HCl buffer at pH 8.0, decreases the LPS
content of the proteins by a factor or about 100, for a total
decrease in the LPS/LPF ratio of between 10,000 and 100,000.
Subsequent washing of the column with further volumes preferably
about 5 volumes of buffer, of 50 mM Tris HCl at pH 8.0, removes the
non-ionic detergent.
The FHA is eluted from the column by contacting the column with
buffer solution, for example, 5 volumes of 50 mM Tris HCl at pH
8.0, containing about 0.1 to about 0.2 mM sodium chloride,
preferably about 0.12 mM. The LPF next is eluted from the column by
contacting the column with buffer solution, for example, 5 volumes
of 50 mM Tris HCl at pH 8.0, containing at least about 0.2M of
sodium chloride, preferably about 0.6M.
The eluted solutions are assayed for protein content. By this
procedure, LPF and FHA recoveries of approximately 60 to 65% and 65
to 70% respectively of the initial contents of these proteins in
the broth have been obtained.
Further purification of LPF and FHA may be effected using a column
of packed hydroxyapatite about 5 to about 8 cm in height and about
5 to about 30 cm in diameter. The column is washed and equilibrated
prior to use. The eluate containing LPF is applied to the column at
a loading of about 0.5 to about 1 mg/ml of packed gel at a linear
flow rate of about 15 to about 25 cm/hr to adsorb the LPF
therefrom.
The column is washed with a suitable buffer, for example, 5 column
volumes of 30 mM potassium phosphate at pH 8.0, following which the
LPF is eluted with about 5 to about 10 column volumes of an eluting
medium, for example, 75 mM potassium phosphate at pH 8.0,
containing about 0.1 to about 0.3M sodium chloride, preferably
about 0.225M.
The procedure may be repeated for the FHA-containing eluant, with
elution being effected using an aqueous elution medium, for
example, 200 mM potassium phosphate at pH 8.0, containing at least
about 0.2M sodium chloride, preferably about 0.6M.
In these hydroxyapatite purification procedures, typical recoveries
of the pure protein are about 80 to about 100% with the respective
proteins having a purity of at least 90%.
Detoxification of the further purified LPF and FHA may be effected
in order to provide these materials in a form suitable for
formulation as a non-toxic vaccine. It is preferred to effect
detoxification of the LPF protein using glutaraldehyde in the
presence of glycerol while it is preferred to effect detoxification
of the FHA protein using formaldehyde in the presence of
glycerol.
The invention is illustrated further by the following Examples.
EXAMPLES
Methods of protein biochemistry, fermentation and assays used but
not explicitly described in this disclosure and these Examples are
amply reported in the scientific literature and are well within
those skilled in the art.
Example 1
This Example illustrates the growth of B.pertussis in
fermentors.
Bordatella pertussis was seeded into a fermentor containing 250 L
of broth (modified Stainer-Scholte medium). During the period of
perfentation, monosodium glutamate (2.18 kg) and the growth
factors, glutathione (41 g), ferrous sulphate (2.7 g), calcium
chloride (5.5 g), ascorbic acid (109 g), niacin (1.1 g) and
cysteine (10.9 g), were added at intervals to increase the yields
of LPF. At the end of a 48 hour fermentation period, the broth was
run through a continuous centrifuge to remove the majority of the
cells. This suspension, which contains both the LPF and FHA in
solution, was further clarified by micro-filtration on cellulose
acetate membranes (0.22 .mu.m pore size). The sterilized filtrate
was concentrated approximately 10-fold using a 20,000 NML membrane
and then assayed for protein by the dye-binding method.
Example 2
This Example illustrates the isolation of LPF and FHA on a number
of different matrices.
A number of 1 milliliter columns were packed with various matrices
and equilibrated with 50 mM Tris HCl at pH 8.0, 10 mM potassium
phosphate at pH 8.0 or water. The matrices included Orange A-, Blue
A-, Green A-, Red A-agaroses, Blue Sepharose, Blue B-, Reactive
Blue 4-, Cibacron Blue 3GA-, Reactive Brown 10-, Reactive Green
19-, Reactive Yellow 86-Sepharose, non-derivatized agarose,
Ultragel ACA44, Sephadex G50, Sepharose 6B, Sepharose CL4B,
S-Sepharose, Q-Sepharose, cellulose sulphate, QAE-cellulose,
CM-cellulose, Perlite and Celite.
B. pertussis culture broth was centrifuged, sterile filtered
through a 0.2 u membrane and concentrated approximately 10 fold by
ultrafiltration on 20 kD NML membranes. Broth concentrates were
diluted with water so that the ionic strength was less than or
equal to 4 mS/cm. Samples between 2 to 10 ml were loaded onto the
columns by gravity feed and then washed with excess 10 mM potassium
phosphate, followed by 50 mM Tris HCl buffer at pH 8.0. Each column
was eluted with 50 mM Tris HCl at pH 8.0 containing either 0.6M or
1.0M sodium chloride. Fractions were analysed by absorbance at 280
nm and on SDS-PAGE. All of the matrices were found to adsorb LPF
and FHA. The eluted LPF and FHA were found to be highly
purified.
In a similar experiment using white quartz sand, a column 1.5 cm in
diameter and 18 cm in height was washed and loaded with the same
diluted broth concentrate to adsorb LPF and FHA therefrom and
washed. The column then was eluted first with 50 mM Tris HCl at pH
8.0 containing 0.1M sodium chloride, followed by Tris buffer
containing 1.0M sodium chloride, so as to elute first the LPF and
then the FHA. The separately eluted LPF and FHA respectively were
found to be highly purified.
Example 3
This Example illustrates the large scale separation of LPF and FHA
using a chromatographic column of Perlite.
The broth concentrate, prepared as described in Example 1, was
diluted with water to a conductivity of approximately 4 mS/cm, such
that the final loading of protein was approximately 3 mg of crude
protein per milliliter of packed Perlite. The packed Perlite column
was 18 cm high and 10 cm in diameter and was prewashed with 1.4 L
of Water for Injection (WFI). The diluted concentrate was applied
to the column at a linear flow rate of 100 cm/hr. The proteins
bound to the Perlite were almost exclusively LPF and FHA with most
of the contaminating protein and lipopolysaccharide (LPS) passing
through. The column was washed with 1.4 L of a buffer containing 50
mM Tris HCl at pH 8.0. A subsequent wash with detergent, composed
of 1.4 L of a 0.5% (v/v) Triton X-100 solution in 50 mM Tris HCl
buffer at pH 8.0, reduced the LPS content by a further factor of
100, for a total reduction in the LPS/LPF ratio of between 10,000
to 100,000. The column then was washed with a further 1.4 L 50 mM
Tris HCl at pH 8.0 to remove the Triton X-100. The LPF then was
eluted from the column with 50 mM Tris HCl at pH 8.0 containing
0.12 mM sodium chloride. The FHA was eluted from the column using
50 mM Tris HCl at pH 8.0 containing 0.6M sodium chloride.
Approximately 1.4 L of each elution buffer was used. The solutions
then were assayed for protein content by the dye-binding assay. LPF
and FHA recoveries were 60% and 65%, respectively, based on ELISA
values.
Example 4
This Example illustrates the batch adsorption of LPF and FHA on
Perlite.
B.pertussis broth concentrates (60 ml) were diluted 4-fold with
water to a conductivity of approximately 4 mS/cm and Perlite (2g)
added. The mixture was rotated slowly at 4.degree. C. for 3 hr. The
mixture was vacuum filtered on a sintered glass filter and the
residual Perlite was rinsed into the filter with 50 mM Tris HCl at
pH 8.0 (20 ml). The Perlite was washed with 4.times.50 ml of the
Tris buffer and then eluted with 3.times.20 ml of 50 mM Tris HCl at
pH 8.0 containing 1.0M sodium chloride. The eluates were pooled and
assayed using an ELISA assay. LPF recoveries were calculated to be
at least 65%.
EXAMPLE 5
This Example illustrates the further purification of LPF on
hydroxyapatite.
Hydroxyapatite was packed into a column 5 to 30 cm diameter and 6
cm height. The column was washed with 200 mM potassium phosphate at
pH 8.0, 1M potassium chloride, 0.5% Triton X-100 and equilibrated
with 10 mM potassium phosphate at pH 8.0 prior to use. The LPF
solution, recovered as described in Example 3, was applied to the
column at a loading of approximately 0.5 mg of protein/ml of packed
gel at a linear flow rate of approximately 20 cm/hr. The column was
washed with 500 ml of 30 mM potassium phosphate at pH 8.0. The LPF
was eluted with 1 L of 75 mM potassium phosphate at pH 8.0
containing 0.225M sodium chloride. The resulting LPF was at least
90% pure. The LPF was assayed for protein by the dye binding
method. The LPF recovery was approximately 90% for this step.
Example 6
This Example illustrates the further purification of FHA on
hydroxyapatite.
The hydroxyapatite was packed and washed in a column of the same
size as detailed in Example 5. The FHA fraction from the Perlite
separation described in Example 3 was applied to the column at a
linear flow rate of 20 cm/hr and a loading of 0.5 mg of protein/ml
of packed gel. The column was washed with 500 ml each of 30 mM
potassium phosphate at pH 8.0, 30 mM potassium phosphate at pH 8.0
containing 0.5% (v/v) of Triton X-100 and 30 mM potassium phosphate
at pH 8.0. Any remaining LPF in the fraction first was eluted with
500 ml of 85 mM potassium phosphate at pH 8.0 and the FHA then was
eluted with 200 mM potassium phosphate at pH 8.0 containing 0.6M
potassium chloride. The resulting FHA was at least 90% pure. The
FHA was assayed for protein by the Lowry method. FHA recovery for
this column was approximately 90%.
Example 7
This Example illustrates the detoxification of LPF with
glutaraldehyde.
The purified LPF, prepared as described in Example 5, in 75 mM
potassium phosphate at pH 8.0 containing 0.22M sodium chloride was
diluted with an equal volume of glycerol to a protein concentration
of approximately 200 .mu.g/ml. The solution was heated to
37.degree. C. and detoxified by the addition of glutaraldehyde to a
final concentration of 0.5% (w/v). The mixture was kept at
37.degree. C. for 4 hr and followed by the addition of aspartic
acid (1.5M) to a final concentration of 0.25M. The mixture was
incubated at room temperature for 1 and then diafiltered with 10
volumes of 10 mM potassium phosphate at pH 8.0 containing 0.15M
sodium chloride to remove both the glycerol and the glutaraldehyde.
The LPF toxoid was sterile filtered through a 0.2 u membrane.
Example 8
This Example illustrates the detoxification of FHA with
formaldehyde.
The purified FHA, prepared as described in Example 6, in 200 mM
potassium phosphate at pH 8.0 containing 0.6M potassium chloride
was diluted with glycerol to give a final concentration of 25% V/V.
The protein concentrations was approximately 500 .mu.g/ml based on
the Lowry protein assay. The FHA solution was heated to 37.degree.
C. and a 1.5M solution of L-lysine HCl at pH 8.0 was added to a
final concentration of 50 mM. Formaldehyde was added to a final
concentration of 0.25% V/V. Detoxification was carried out at
37.degree. C. for a period of 6 weeks. The resulting toxoid was
diafiltered against 10 volumes of 10 mM potassium phosphate at pH
8.0 containing 0.5M sodium chloride to remove both the glycerol and
the formaldehyde. The toxoid solution was sterile filtered through
a 0.2 .mu. membrane.
Example 9
This Example illustrates the use of detoxified LPF and FHA in
producing protective antibodies.
Guinea pigs (SPF) were prescreened for pertussis antibody titres,
and only those animals which showed low background titres were used
in the experiment.
Animals were injected with 0.5 ml of test material at day zero.
Test materials employed in the tests were the purified and
detoxified LPF and FHA products produced by the procedures of
Examples 7 and 8 respectively ("adsorbed"), LPF and FHA isolated
from broth but not processed by the invention ("unadsorbed") and
conventional whole cell vaccine.
Four weeks after injection, the animals were bled and the sera
tested for PT and FHA antibodies by ELISA. Sera also were tested
for CHO antitoxin activity. At day 35, the animals were boosted
with the same dose of antigen and finally the animals were bled at
day 49 and the sera tested. The results are shown in the following
Table I:
TABLE I
__________________________________________________________________________
IMMUNOGENICITY OF PERTUSSIS TOXOID In guinea pigs at 25 ug dose
ELISA .times. 10.sup.-3 CHO LPF FHA Protein Units 1st 2nd 1st 2nd
ug 1st 2nd bleed bleed bleed bleed
__________________________________________________________________________
LPF (unadsorbed) 25 14 640 3 256 (adsorbed) 25 433 1664 59 410 FHA
(unadsorbed) 25 52 33 (adsorbed) 25 14 205 Whole Cell human 4 30 2
21 4 21 (unadsorbed) dose
__________________________________________________________________________
All results are reciprocal reactive titres.
The results set forth in the Table indicate that when compared to
the conventional whole cell vaccine and unprocessed LPF and FHA
proteins, the purified and detoxified LPF and FHA proteins provided
by the procedures of the invention give considerably higher
antibody titres.
Example 10
This Example illustrates the use of the purified antigens in the
mouse protection test.
Taconic mice (15 to 17g) were injected at day zero with 0.5 ml of
the test sample intraperitoneally, in three doses. Each dose was
injected into 16 mice. At day 14, the mice were challenged with an
intracerebral injection of a standard does of B.pertussis. Control
mice also were injected at the same time to ascertain the
effectiveness of the challenge. Three days after the challenge, the
number of animal deaths was recorded every day up to and including
day 28. At day 28, paralysed mice and mice with cerebral edema also
were recorded as dead.
Results were recorded as ED.sub.50, which is the dose at which half
the mice survive the challenge. This was done using a computer
programme after plotting the survivors divided by the total number
of mice in each category at each dose.
The result of this experiment showed that the ED.sub.50 of a
mixture of LPF and FHA was less than [lug LPF+2 ug FHA], and thus a
mixture of the two purified proteins was protective against the
disease.
SUMMARY OF DISCLOSURE
In summary of this disclosure, the present invention provides a
novel and unexpected method for the separation of proteins from the
growth media of B.pertussis that can be used as antigens to elicit
protection against the disease of pertussis. The novel method
employs a difference in ionic strengths of the solutions from which
the proteins are adsorbed and the solutions used to desorb them
from the substrate. A further aspect of the invention is the
reduction of LPS by washing the adsorbed proteins with a solution
of detergent. The use of glycerol or sucrose for preventing protein
aggregation during the detoxification process, is an important
aspect of the invention since protein aggregation could result in
up to 95% of protein losses at the final step of the process.
Modifications are possible within the scope of this invention.
* * * * *